Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Reaction of selenomethionine with o-benzoquinone

A new reaction of selenomethionine with o-benzoquinone is described. This reaction can be used to distinguish between methionine and selenomethionine.     

Incorporation of the sulfur of L-[35S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Metabolism of 5′-methylthioadenosine in Aspergillus nidulans an alternative pathway for methionine synthesis via utilization of the nucleoside methylthio group

Experiments in which 5′-methylthioadenosine was used as a culture supplement for methionine-requiring mutants of Aspergillus nidulans with various enzymatic lesions indicated that the methylthio group derived from the nucleoside can be recycled to methionine. The results strongly suggest that methionine may be synthesized in the reaction catalyzed by homocysteine synthase (EC 4.2.99.10) in which O-acetylhomoserine is an acceptor of the methylthio group. The first step on the salvage pathway of the methylthio group is, in Aspergillus nidulans, phosphorolytic cleavage of 5′-methylthioadenosine to adenine and 5-methylthioribose 1-phosphate catalyzed by a specific phosphorylase.     

The regulation of one-carbon oxidation in the rat by nitrous oxide and methionine

Formate is oxidized to CO₂ in the rat by folate-dependent reactions. Nitrous oxide treatment inhibited hepatic methionine synthetase activity, reduced hepatic S-adenosyl-l-methionine (Ado-Met) and tetrahydrofolate (H₄ folate) concentrations and decreased the rate of formate oxidation in the rat. The administration of methionine to nitrous oxide-treated rats increased hepatic Ado-Met concentrations and restored hepatic H₄folate levels and formate oxidation to control values but did not reverse the inhibition of methionine synthetase. Positive correlations were observed between hepatic Ado-Met levels and H₄folate concentrations and between hepatic H₄folate concentrations and formate oxidation. These results suggest that alterations in hepatic H₄folate concentrations may profoundly influence the oxidation of one-carbon compounds. They confirm the importance of the methionine synthetase reaction as a major source of regeneration of H₄folate. These findings also indicate that methionine acts at a site other than the methionine synthetase reaction to restore hepatic H₄folate concentrations and formate oxidation to control values in nitrous oxide-treated rats.     

Regulation of mitochondrial glycine decarboxylase from pea mitochondria

Reactions of glycine cleavage were assayed in mitochondria isolated from cotyledons of germinating pea seeds. These reactions, which included the exchange of bicarbonate with C-1 of glycine and an NAD-stimulated decarboxylation of glycine, were maximal under aerobic conditions at pH 7·8. The apparent Michaelis-Menten constants for glycine and bicarbonate in the exchange reaction were 1·8 and 12·5 mM respectively. The K_m for NAD in the decarboxylation reaction was 47 μM. Maximal enzyme activity was observed when mitochon-drial integrity was maintained. Up to 40% inhibition of the decarboxylation reaction was observed when NADH, NADPH or l-methionine were added to the reaction system. When glycine-[2-¹⁴C] was incubated with the isolated mitochondria, labelled CO₂ was evolved in nanomolar quantities. It is concluded that glycine decarboxylase may be of importance in supplying C-1 units for the de novo synthesis of methionine in pea mitochondria.     

Naphthaquinone biosynthesis in moulds: the mechanism for formation of javanicin

1. The following compounds, added to the growth medium of Fusarium javanicum, were converted into labelled javanicin with the percentage incorporations noted in parentheses: [Me-¹⁴C]methionine (0·83); [1-¹⁴C]acetate (0·70); [2-¹⁴C]malonate (0·07). 2. Labelled samples of javanicin were degraded by Zeisel reaction, Kuhn–Roth oxidation and reaction with sodium hypoiodite; acetic acid obtained from the Kuhn–Roth reaction was further degraded by the Schmidt reaction. Labelled methionine was used only for the formation of the methoxyl group, and the remaining carbon atoms were derived by the acetate-plus-polymalonate pathway. The methyl group attached directly to the naphthaquinone ring is derived by the reduction of a carboxyl group. 3. The demonstration of this biosynthetic pathway supports the assignment of the methoxyl group at position 7.     

Folic acid and the methylation of homocysteine by Bacillus subtilis

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C₁ transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B₁₂. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.     

MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila

To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.     

Endo R DpnI restriction of Escherichia coli DNA synthesized in vitro. Evidence that the ends of Okazaki pieces are determined by template deoxynucleotide sequence.

A restriction enzyme from Diplococcus pneumoniae, Endo R DpnI, cuts methylated DNA on cellophane discs into pieces which are about the same size as Okazaki pieces.DNA was synthesized in vitro on cellophane discs in the presence of β-nicotinamide mononucleotide to prevent joining of Okazaki pieces. This DNA was methylated by the addition of S-adenosyl methionine to the reaction mixture. When Endo R DpnI was used to cut methylated DNA made in vitro in the presence of S-adenosyl methionine and β-nicotinamide mononucleotide, no decrease in sedimentation of the Okazaki pieces was observed. Control experiments demonstrated that Okazaki pieces were methylated in vitro and that Endo R DpnI was capable of cutting double-stranded DNA containing methylated Okazaki pieces, that is, synthesized in β-nicotinamide mononucleotide and S-adenosyl methionine.These results are interpreted to mean that the ends of Okazaki pieces are non-randomly distributed with respect to 6-methyl adenine residues.     

Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : II. TIME COURSE OF LEVELS OF INTERMEDIATES AND IN VIVO CONVERSION RATES

Ethylene production was stimulated severalfold during the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV). Exogenous methionine or S-adenosylmethionine (SAM) did not increase ethylene evolution from healthy or TMV-infected leaf discs, although both precursors were directly available for ethylene production. This indicates that ethylene production is not controlled at the level of methionine concentration or availability, nor at the level of SAM production or concentration. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) stimulated ethylene production considerably. Thus, ethylene production is primarily limited at the level of ACC production.     

Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid

Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants

Background

Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.

Results

The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 μmol/min/mg protein and the Km for methionine and ATP was 288 μM and 76 μM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 μM, while the MIC for 8-azaguanine was >1.0 mM.

Conclusion

The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.     

Structural plasticity of the thioredoxin recognition site of yeast methionine S-sulfoxide reductase Mxr1

The methionine S-sulfoxide reductase MsrA catalyzes the reduction of methionine sulfoxide, a ubiquitous reaction depending on the thioredoxin system. To investigate interactions between MsrA and thioredoxin (Trx), we determined the crystal structures of yeast MsrA/Mxr1 in their reduced, oxidized, and Trx2-complexed forms, at 2.03, 1.90, and 2.70 Å, respectively. Comparative structure analysis revealed significant conformational changes of the three loops, which form a plastic “cushion” to harbor the electron donor Trx2. The flexible C-terminal loop enabled Mxr1 to access the methionine sulfoxide on various protein substrates. Moreover, the plasticity of the Trx binding site on Mxr1 provides structural insights into the recognition of diverse substrates by a universal catalytic motif of Trx.     

Biosynthesis of adenosyl-d-methionine and adenosyl-2-methylmethionine by Candida utilis

Supplementation of the culture medium of Candida utilis with d-methionine or 2-methyl-dl-methionine leads to intracellular synthesis of S-adenosyl-d-methionine and S-adenosyl-2-methylmethionine. The identity of the sulfonium compounds was established by tracer technique, chromatography, acid hydrolysis, and examination of the released methionine and 2-methylmethionine. In addition to the expected sulfur amino acid component, both adenosine sulfonium fractions contained S-adenosyl-l-methionine. This is explained by transmethylation of S-adenosyl-d-methionine and of S-adenosyl-2-methyl-methionine with endogenous l-homocysteine; the resulting l-methionine reacts with ATP to form S-adenosyl-l-methionine. Experiments with purified cell-free preparations of S-adenosylmethionine synthetase (EC 2.5.1.6) from C. utilis confirmed the reaction of ATP with d-methionine or 2-methyl-dl-methionine.     

Methionine analogs inhibit production of β-subunit of soybean 7S protein

We have previously reported that exogenous methionine inhibits production of the β-subunit of the 7S storage protein in cultured soybean cotyledons, and that this inhibition involves lack of functional mRNA for the β-subunit. Analogs of methionine were used to study this inhibition. Cycloleucine, norleucine, norvaline and S-ethylcysteine treatments prevented accumulation of the β-subunit. The effects of cycloleucine and norleucine on β-subunit synthesis might have been indirect, since these compounds inhibited growth and caused a 2- to 3-fold increase in free methionine concentration. Norvaline did not affect free methionine concentration, but it did inhibit growth. Treatment with a combination of S-ethylcysteine and aminoethoxyvinylglycine prevented appearance of the β-subunit without inhibiting growth or raising the S-adenosylmethionine concentration. Thus, accumulation of S-adenosylmethionine does not appear to mediate the effect of exogenous methionine on β-subunit production. Treatment with S-ethylcysteine raised free methionine concentration only 34%, so S-ethylcysteine was probably acting directly to inhibit β-subunit production. Measurements of free methionine concentrations in seeds of different sizes, taken from intact plants, suggested that the relatively late appearance of the β-subunit in normal soybean seed development may be due to the presence of high levels of free methionine in very young seeds.     

The purification of methionine sulfoxide reductase from Escherichia coli

A sensitive assay, based on the acylation of tRNA^Met, has been developed to measure the enzymatic reduction of methionine sulfoxide to methionine. Using this assay, methionine sulfoxide reductase has been purified to near homogeneity from extracts of Escherichia coli.